Xylose reductase mutants with improved specificity towards xylose and their uses are described.
Xylitol (1) is a pentitol and is used not only as a sweetener but also as a platform chemical for the production of industrially important chemicals. Studies have shown that among sugar substitutes, xylitol is one of the most promising candidates for application in a wide range of products due to several favorable properties. These include anti-cariogenicity, suitability for use by diabetic patients, and good gastrointestinal tolerance, in addition to possibly preventing osteoporosis and ear infections. In spite of its advantages, the use of xylitol is currently limited and falls well short of another, cheaper sugar alternative, sorbitol in the billion dollar polyol market. Other than its use as a sweetener, xylitol is also an industrially important chemical, and the US Department of Energy (DOE) has named it among one of their top 12 platform chemicals from agricultural sources.
Xylose reductase (XR) is an enzyme found commonly in yeast and fungal organisms often with several isozymes in the same species. This enzyme catalyzes the first step in the metabolism of D-xylose and other pentose sugars by reducing the linear aldehyde form of the sugar to xylitol (or a corresponding sugar alcohol). Xylitol can then be oxidized to xylulose by NAD-dependent xylitol dehydrogenase and phosphorylated by D-xylulokinase. The resulting sugar phosphate can enter the pentose phosphate pathway. The reversible reduction of xylitol by XR occurs concomitantly with NAD(P)H oxidation. In general, XR is specific for NADPH, but in some cases it utilizes both NADPH and NADH and in at least one case prefers NADH over NADPH. The different forms of XR in the same species usually have different cofactor preferences and they are likely needed to maintain the redox balance between nicotinamide cofactors under a variety of growth conditions. In order to maintain this balance under anaerobic conditions, XR is likely to be NADH-dependent because the enzyme in the following step (xylitol dehydrogenase) is NAD specific. However, under aerobic conditions either cofactor can be used since cofactors can be regenerated. Some yeast species have solved this problem by utilizing one form of XR with dual cofactor specificity.
Commercially available xylitol is obtained by processing its oxidized form, the pentose D-xylose. Second only to glucose, xylose is the most common sugar in nature, and is the primary component of plant hemicellulose. Unlike cellulose, which is a homogenous glucose polymer, hemicelluloses are complex polymers of several sugars (D-xylose, L-arabinose, D-glucose, D-mannose, and D-galactose, etc.) and sugar acids. Xylose is purified from pretreated hemicellulose and then chemically reduced to xylitol at high pressure (40 atm), high temperature (135° C.) with elemental hydrogen over a carcinogenic Raney-Nickel catalyst. Recent studies have tried to formulate several safer and environmentally friendlier techniques based on biotechnology to produce xylitol using a xylose reductase enzyme (XR). However, the techniques previously described require the use of purified xylose, due to the promiscuous nature of XRs toward sugars found in hemicellulose. Separating xylose from arabinose is particularly difficult, being epimers and having the same molecular weight. In addition all known catalysts, whether enzymatic XRs or synthetic Raney-Nickel, can reduce both sugars efficiently.
One alternative to purifying xylose from impurities is to engineer an XR to preferentially utilize xylose, or more simply, to engineer an XR to accept arabinose poorly compared to xylose. Such an enzyme would negate the need for extensive purification of xylose prior to reduction, increasing yield and simultaneously decreasing production costs.